Toll-like receptors in central nervous system injury and disease: A focus on the spinal cord

Brain, Behavior, and Immunity xxx (2014) xxx–xxx

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Brain, Behavior, and Immunity journal homepage: www.elsevier.com/locate/ybrbi

Full-Length Review

Toll-like receptors in central nervous system injury and disease: A focus on the spinal cord Adee Heiman a,1,2, Alexandra Pallottie a,b,1, Robert F. Heary a, Stella Elkabes a,⇑ a b

Reynolds Family Spine Laboratory, Department of Neurological Surgery, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, United States Graduate School of Biomedical Sciences, New Jersey Medical School, Rutgers, The State University of New Jersey, Newark, NJ, United States

a r t i c l e

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Article history: Received 13 March 2014 Received in revised form 17 June 2014 Accepted 28 June 2014 Available online xxxx Keywords: Innate immunity Gliosis Motor neuron Regeneration Neuroprotection Cytokine Chemokine Neuropathic pain Amyotrophic lateral sclerosis Spinal cord injury

a b s t r a c t Toll-like receptors (TLRs) are best known for recognizing pathogens and initiating an innate immune response to protect the host. However, they also detect tissue damage and induce sterile inflammation upon the binding of endogenous ligands released by stressed or injured cells. In addition to immune system-related cells, TLRs have been identified in central nervous system (CNS) neurons and glial subtypes including microglia, astrocytes and oligodendrocytes. Direct and indirect effects of TLR ligands on neurons and glial subtypes have been documented in vitro. Likewise, the effects of TLR ligands have been demonstrated in vivo using animal models of CNS trauma and disease including spinal cord injury (SCI), amyotrophic lateral sclerosis (ALS) and neuropathic pain. The indirect effects are most likely mediated via microglia or immune system cells that infiltrate the diseased or injured CNS. Despite considerable progress over the past decade, the role of TLRs in the physiological and pathological function of the spinal cord remains inadequately defined. Published reports collectively highlight TLRs as promising targets for therapeutic interventions in spinal cord pathology. The findings also underscore the complexity of TLR-mediated mechanisms and the necessity for further research in this field. The goals of the current review are to recapitulate the studies that investigated the role of TLRs in the spinal cord, to discuss potential future research directions, and to examine some of the challenges associated with pre-clinical studies pertinent to TLRs in the injured or diseased spinal cord. Ó 2014 Elsevier Inc. All rights reserved.

1. Introduction Toll-like receptors (TLRs) are the mammalian homologues of the Drosophila melanogaster Toll (Medzhitov et al., 1997), a transmembrane receptor (Hashimoto et al., 1988) that plays a crucial role in mediating Drosophila immunity (Halfon et al., 1995; Lemaitre et al., 1996; Qiu et al., 1998) as well as embryogenesis, particularly in the establishment of the dorsal–ventral axis and motoneuron development (Anderson et al., 1985a,b). Toll-like receptors are one of several classes of pattern-recognition receptors (PRRs), which are collectively known for their role in the activation of the innate immune system and the subsequent orchestration of the adaptive immune response (for reviews see Kaisho and Akira, 2006; Medzhitov and Janeway, 2000). Upon

⇑ Corresponding author. Address: New Jersey Medical School, 205 South Orange Avenue, Cancer Center F-1204, Newark, NJ 07103, United States. E-mail address: [email protected] (S. Elkabes). 1 Equal contribution. 2 Current address: University of Louisville School of Medicine, HSC Instructional Building, Louisville, KY 40292, United States.

recognition of conserved molecular motifs expressed by pathogens, referred to as ‘‘pathogen-associated molecular patterns’’ (PAMPs), TLRs initiate a cascade of intracellular events involving the Nuclear Factor-kappa B (NF-jB)-dependent production and release of cytokines and chemokines (Hirschfeld et al., 1999; Medzhitov et al., 1997). PAMPs consist of signature sequences crucial for pathogen survival in the host and comprise all macromolecular classes including lipopolysaccharide (LPS), a component of the cell wall of gram negative bacteria (Poltorak et al., 1998a), peptidoglycans derived from gram positive bacterial cell walls (Takeuchi et al., 1999a), flagellin (Hayashi et al., 2001), lipoproteins (Brightbill et al., 1999), as well as single stranded RNA (Diebold et al., 2004; Heil et al., 2004) or double stranded RNA (Alexopoulou et al., 2001; Liu et al., 2008) and unmethylated CpG DNA (Hemmi et al., 2000; Latz et al., 2007). Thus far, TLRs 1–10 have been identified in humans (Chaudhary et al., 1998; Chuang and Ulevitch, 2000, 2001; Rock et al., 1998; Takeuchi et al., 1999b) with TLR11 rendered non-functional by the presence of a stop codon (Zhang et al., 2004). TLRs 1–9 and TLR 11–13 have been identified in mice (Du et al., 2000; Hemmi et al., 2000; Poltorak et al., 1998a,b; Sebastiani et al., 2000; Shi

http://dx.doi.org/10.1016/j.bbi.2014.06.203 0889-1591/Ó 2014 Elsevier Inc. All rights reserved.

Please cite this article in press as: Heiman, A., et al. Toll-like receptors in central nervous system injury and disease: A focus on the spinal cord. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.06.203

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A. Heiman et al. / Brain, Behavior, and Immunity xxx (2014) xxx–xxx

et al., 2011a; Takeuchi et al., 1999b) with TLR10 rendered nonfunctional by a retroviral insertion (Hasan et al., 2005). TLRs 1, 2, 4–6, 10–12 are located on the cell surface whereas TLRs 3, 7–9, and 13 are localized to the membranes of endosomal compartments and the endoplasmic reticulum in immune system-related cells including polymorphonuclear leukocytes (PMNs; Muzio et al., 2000), monocytes (Kadowaki et al., 2001; Sabroe et al., 2002), dendritic cells (Kadowaki et al., 2001), T lymphocytes (Muzio et al., 2000) and B lymphocytes (Bourke et al., 2003; Hornung et al., 2002). However, evidence indicates that TLRs are also expressed in the human (Bsibsi et al., 2002) and rodent (Laflamme et al., 2001) central nervous system (CNS) cells, including microglia (Bsibsi et al., 2002; Kigerl et al., 2007), oligodendrocytes (Bsibsi et al., 2002; Kigerl et al., 2007), astrocytes (Bsibsi et al., 2002; Bowman et al., 2003; Kigerl et al., 2007) and neurons (David et al., 2013; Lafon et al., 2006; Ma et al., 2006). Importantly, TLRs also play a role in non-infectious conditions associated with tissue insult and repair wherein endogenous ligands known as ‘‘damage-associated molecular patterns’’ (DAMPs) may be released in response to cellular stress, injury or death. DAMPs can bind and trigger TLR activation and consequently contribute to sterile inflammation. Some DAMPs are confined to the intracellular space under physiological conditions and are released into the extracellular space following injury- or disease-induced cellular damage and death. These DAMPs include high mobility group box 1 (HMGB-1) (Park et al., 2004), heat shock proteins (HSPs) (Asea et al., 2002; Ohashi et al., 2000), microRNA (Lehmann et al., 2012), mitochondrial RNA and DNA (Kariko et al., 2005; Zhang et al., 2010) and histones (Huang et al., 2011). In addition, stressed and injured cells release activated proteases which degrade the extracellular matrix, and thereby, generate additional DAMPs including hyaluronic acid (Termeer et al., 2002), fibrinogen (Smiley et al., 2001), fibronectin (Gondokaryono et al., 2007) and biglycan (Schaefer et al., 2005). TLRs have been implicated in neuroinflammation associated with a number of neurological and neurodegenerative conditions of the CNS. Depending on the cell type and the condition under which the receptor is activated, both detrimental and beneficial roles have been attributed. The goal of this review is to highlight the expression and the roles of TLRs in the healthy spinal cord and in pathological conditions that affect the spinal cord including traumatic injury, neuropathic pain and amyotrophic lateral sclerosis (ALS).

2. Overview of toll-like receptor signaling TLRs are type I transmembrane glycoproteins containing a leucine-rich repeat (LRR; Hashimoto et al., 1988) motif on the extracellular domain that mediates ligand binding, and a toll-interleukin-1 (IL-1) receptor (TIR) intracellular domain (Rock et al., 1998) that facilitates the binding of downstream adaptor proteins (Medzhitov et al., 1998; Muzio et al., 1998; Takeuchi et al., 2000). Following ligand binding, TLRs form homodimers (Bovijn et al., 2012) or heterodimers (Hajjar et al., 2001; Ozinsky et al., 2000a,b) and initiate signaling through either the myeloid differentiation 88 (MyD88)-dependent or MyD88-independent pathways (Akira and Takeda, 2004). Toll-like receptors 1–2 and 5–13 signal primarily through the MyD88 protein. In addition, TLR2 uses the bridging adaptor toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) to recruit MyD88 to its TIR domain (Yamamoto et al., 2002). TLRs that primarily utilize the MyD88dependent pathway can activate the transcription factors activator protein (AP)-1 (Chiu et al., 2009; Liu et al., 2009a) via the p38/c-Jun N-terminal kinase (JNK)/extracellular signal regulated kinase (ERK) pathway (An et al., 2002), Nuclear Factor-jB (NF-jB; Zhang et al.,

1999) or interferon regulatory factor 5 (IRF5; Takaoka et al., 2005). Translocation of these transcription factors to the nucleus promotes the transcription of inflammatory cytokines. Toll-like receptor 3 signals exclusively through the MyD88independent, TIR-domain-containing adaptor protein inducing IFN-b (TRIF) pathway which drives the activation of interferon regulatory factor 3 (IRF3; Sato et al., 2003) and IRF7 (Fitzgerald et al., 2003; Han et al., 2004), resulting in transcription of type I interferons (IFNs; Sato et al., 1998a,b). In addition, TLR3 can activate the transcription factors IRF5 (Barnes et al., 2001; Takaoka et al., 2005), AP-1 (Chiu et al., 2009; Liu et al., 2009a) and NF-kB through the adaptor protein TNF receptor associated factor 6 (TRAF6) leading to transcription of inflammatory cytokines (Mukundan et al., 2005). TLR4 is unique as it can signal through both MyD88 (Kawai et al., 1999; Takeuchi et al., 2000) and TRIF signaling pathways (Fitzgerald et al., 2003; Yamamoto et al., 2003). Some of the major TLR signaling pathways have been illustrated in Fig. 1 and the current knowledge about TLR signaling has been discussed, in detail, in a recent review (Sasai and Yamamoto, 2013).

3. Inflammation and tissue damage elicited by intrathecal or intraspinal delivery of TLR agonists to naïve rodents Toll-like receptors 1–10 are present in the healthy adult human spinal cord with TLR2, TLR4, TLR6, and TLR7 showing the highest expression at the mRNA level (Nishimura and Naito, 2005). TLR mRNA (Adhikary et al., 2011; Kigerl et al., 2007) or protein (David et al., 2013) has also been detected in the rodent spinal cord. A number of studies investigated the molecular and cellular responses in the spinal cord following intrathecal (i.t.) or intraspinal (direct injection into the spinal cord parenchyma) delivery of TLR ligands to naïve rodents. Intraspinal injection of zymosan, a TLR2 agonist, activated resident microglia and induced infiltration of monocytes and blood-derived macrophages, which was paralleled by demyelination, axonal injury, and astroglial activation (Popovich et al., 2002). However, despite these histopathological changes, only a small portion of the injected rats developed transient anomalies in open field locomotor function (Popovich et al., 2002). The detrimental effects of intraspinal zymosan on myelin and axonal integrity have been attributed to TLR2-mediated activation of macrophages (Schonberg et al., 2007). Whereas the studies of Schonberg et al. (2007) on the spinal cord suggested that zymosan reduces myelin integrity via the release of toxic effectors by inflammatory cells, investigations on cultured brain oligodendrocytes and oligodendrocyte progenitor cells (OPCs) indicated that zymosan can have direct and beneficial effects on these cells (Bsibsi et al., 2012). Zymosan promoted the survival and differentiation of brain OPCs and immature oligodendrocytes, in vitro, without altering the survival of mature oligodendrocytes (Bsibsi et al., 2012). In contrast to zymosan, hyaluronan, another TLR2 agonist, blocked the maturation of brain OPCs, in vitro (Sloane et al., 2010). The presence of TLR2 immunoreactivity in oligodendrocytes of normal and pathological human brain tissue has been reported (Bsibsi et al., 2002; Sloane et al., 2010) and supports the notion that TLR2 ligands could directly act on cells of the oligodendrocyte lineage, not only in vitro but also in vivo. Based on the aforementioned investigations, we postulate that zymosan could have divergent effects on cells of the oligodendrocyte lineage. Such effects could be mediated through direct as opposed to indirect mechanisms. It is possible that the direct and beneficial effects of zymosan on OPCs and immature oligodendrocytes are detectable only when the cells are isolated from their

Please cite this article in press as: Heiman, A., et al. Toll-like receptors in central nervous system injury and disease: A focus on the spinal cord. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.06.203

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Fig. 1. General schematic of some of the major TLR signaling pathways. Toll like receptors 5, 10–12, TLR1/2 and TLR2/6 heterodimers and TLR 7, 8, and 9 signal through the MyD88-dependent pathway. TLR3 signals through the MyD88-independent pathway. Following ligand binding and the subsequent receptor homo- or heterodimerization the adaptor protein MyD88 is recruited to the TIR domain of each receptor. MyD88 associates with IRAK4, a serine/threonine kinase, which phosphorylates IRAK-1 or IRAK-2. This induces association with TRAF6 which interacts with the TAK1/TAB1/TAB2/TAB3 complex. TAK1 phosphorylates NEMO/IKKa/IKKb, which, in turn, phosphorylates IKB. This facilitates the translocation of NF-jB (comprised of the subunits p50 and p65) to the nucleus where it activates transcription of inflammatory cytokines. TAK1 activation is also linked to the MAPK pathway whereby MAPKK3/6, MAPKK 4/7, and MEK 1/2 induce activation of p38, JNK, and ERK 1/2, respectively. These pathways lead to the translocation of AP-1 to the nucleus and the transcription of inflammatory cytokines. TLR3 utilizes the MyD88-independent pathway and recruits the adaptor protein TRIF. TRIF interacts with TRAF3 which leads to the translocation of IRF7 and IRF3 dimers to the nucleus and transcription of Type I interferons. TLR3 can also signal through TRAF6 which results in the translocation of IRF5 to the nucleus and cytokine transcription. TLR4 is unique in that it can signal through either the MyD88-dependent or MyD88independent pathways. However, because TLR4 cannot interact with MyD88 or TRIF directly, bridging adaptors TIRAP and TRAM, respectively, are required for their recruitment to the TIR domain and subsequent induction of downstream signaling. Similarly, TLR2 requires the bridging adaptor TIRAP in order to recruit MyD88. Abbreviations: AP-1, activator protein; ERK, extracellular signal-regulated kinase; IKB, inhibitor of NF-jB; IKK, inhibitor of NF-jB kinase; IRAK, IL-1 receptor-associated kinase; IRF, interferon regulatory factor; JNK, c-Jun N-terminal kinase; MAPKK, MAP kinase kinase; MEK, mitogen-activated protein kinase; MyD88, myeloid differentiation primary response 88; NEMO, NF-jB essential modulator; NF-jB, Nuclear Factor kappa-light-chain-enhancer of activated B cells; TAB, TAK1-binding protein; TAK, transforming growth factor b (TGF-b)-activated kinase; TBK, TANK-binding kinase; TIRAP, toll-interleukin 1 receptor (TIR) domain containing adaptor protein; TRAF, TNF receptor associated factor; TRAM, TRIF-related adaptor molecule; TRIF, TIR-domain-containing adapter-inducing interferon-b.

milieu and grown in culture. In vivo, the supportive, direct action of zymosan on oligodendrocytes or OPCs could be potentially masked by the presence of neighboring cells, such as macrophages, which release toxic mediators in response to zymosan. Since the studies of Bsibsi et al. (2012) were performed on brain oligodendrocyte cultures, further investigations are necessary to determine whether zymosan exerts beneficial effects on spinal cord OPCs and immature oligodendrocytes both in vitro and in vivo. It is also worth noting that the investigations of Bsibsi et al. (2012) and Sloane et al. (2010), taken together, indicate that opposing

responses can be elicited by distinct agonists of TLR2 acting directly on OPCs, in vitro. Gensel et al. (2009) reported that zymosan-stimulated macrophages play dual and opposing roles in the rat spinal cord. In this study, green fluorescent protein positive dorsal root ganglia (DRG) neurons were transplanted to the cervical spinal cord and intraspinal injections of zymosan were performed at the time of transplantation, in a region caudal to the transplantation site. When zymosan was injected 4 mm caudal to the transplanted neurons, it enhanced growth of DRG axons towards the foci of

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macrophage activation. In contrast, when the caudal zymosan injections were performed closer to the transplanted neurons, it abrogated axonal growth and reduced the viability of the transplanted neurons. In vitro investigations indicated that conditioned medium obtained from zymosan-treated macrophages initially exert a growth-promoting effect on cultured DRG neurons which was followed by neurotoxicity at a later stage (Gensel et al., 2009). The precise mechanisms underlying the dual effects of macrophages have not been identified. Further studies are necessary to define how the initial growth-eliciting effects of macrophage conditioned-medium on DRG neurons are subsequently followed by neuronal toxicity, in vitro. In addition, it remains to be determined whether similar results can be obtained in neuron-macrophage co-cultures, which allow the cross-talk between the two cell populations. Moreover, the influence of other spinal cord cells on macrophages or transplanted neurons, in the experimental paradigm used by Gensel et al. (2009), requires further investigations. The studies of Felts et al. (2005) indicated that intraspinal injection of LPS, the TLR4 agonist, increases inducible nitric oxide synthase (iNOS) expression in microglia/macrophages, elevates IL-1b expression and promotes infiltration of immune system-related cells including PMNs and T-lymphocytes. This focal inflammatory response was associated with transient demyelination, axonal loss and astroglial activation (Felts et al., 2005). In addition, Schonberg et al. (2007) reported an apparent reduction in myelin in the lesions formed in response to an intraspinal injection of LPS. In this study, occasional demyelinated axons and robust macrophage activation were also observed. Interestingly, LPS promoted the proliferation of Neural/Glial antigen 2 (NG2) immunopositive OPCs and their differentiation into mature oligodendrocytes. This beneficial effect of LPS was attributed to an increase in ciliary neurotrophic factor (CNTF) expression in the lesion milieu (Schonberg et al., 2007). Of note, earlier investigations had shown that CNTF promoted the survival (Louis et al., 1993; Mayer et al., 1994) and differentiation (Mayer et al., 1994) of oligodendrocytes. Less is known about the outcomes of TLRs 3, 7, 8, and 9 activation, inhibition or deletion in the spinal cord. Intrathecal administration of CpG ODN 1826, a TLR9 agonist, to female mice increases Tumor Necrosis Factor-a (TNF-a), IL-1b and Chemokine (C-X-C motif) ligand 1 (CXCL1) mRNA expression and elicits an elevation in the number of cells of myeloid and hematopoietic origin (David et al., 2013). While TLR3 deficiency impairs synaptic transmission and long-term potentiation in dorsal horn neurons, TLR7 deficiency does not influence these parameters (Liu et al., 2012).

an increase in the expression of TLRs and their downstream effectors (Kigerl et al., 2007). Evidence for the up-regulation of TLR2 and TLR4 mRNA expression in the moderately contused mouse spinal cord was obtained by microarray analysis, in situ hybridization and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). Using laser capture microdissection of individual glial subtypes followed by qRT-PCR, TLR4 expression was primarily localized to CNS macrophages, whereas TLR2 was expressed by both CNS macrophages and astrocytes in equivalent amounts. Up-regulation of TLR1, TLR5, TLR6, TLR7, and TLR9 as well as the TLR4 co-receptor MD-2, and the TLR2 and TLR4 co-receptor CD14 was also observed (Kigerl et al., 2007). Investigations on the rat spinal cord indicated an injury-elicited increase in TLR2 and TLR4 transcript (Adhikary et al., 2011) and protein (Chen et al., 2011) levels, and an up-regulation of TLR1, TLR6 and TLR7 mRNA expression (Adhikary et al., 2011). In rats sustaining a compression SCI, gene expression profiling showed an increase in TLR expression that persisted into the chronic stages, albeit less pronounced than in the acute phase (Chamankhah et al., 2013). 4.2. Functional and cellular responses to spinal cord injury in TLR deficient or mutant mice

Traumatic spinal cord injury (SCI) is characterized by a disruption of the ascending and descending axonal tracts connecting the brain and spinal cord, which can result in locomotor, sensory, and autonomic deficits. The primary mechanical insult is followed by numerous molecular and cellular changes that result in edema, breakdown of the blood–spinal cord barrier, activation of resident glial cells, infiltration of peripheral immune cells, release of proinflammatory effectors including cytokines and chemokines and oxidative stress. As a consequence of these alterations, the spared tissue becomes vulnerable to secondary damage which can lead to cell death, loss of white matter and increased lesion volume. In addition, an astrocytic scar forms around the lesion which constitutes a chemical and physical barrier to the regeneration of transected axons (reviewed in Hausmann, 2003).

In order to explore the role of TLRs in SCI, whole-animal knockout, or mutant, mouse models have been used. In TLR4 mutant mice sustaining a SCI, demyelination, astrogliosis and macrophage activation were more pronounced than in injured, wild type (WT) controls although IL-1b levels were lower than in WT mice (Kigerl et al., 2007). Whereas the evaluation of locomotor function by the main Basso Mouse Scale (BMS) did not reveal any differences between the two groups, computerized gait analysis using the CatWalk apparatus indicated that gait and coordination deficits were significantly more pronounced in TLR4 mutant mice than in WT controls. Similarly, in the same study, TLR2 / mice and WT controls did not show differences in the main BMS scores. However, compared to the WT, TLR2 / mice exhibited sustained deficits in coordination. While total myelin sparing in the injured TLR2 / and WT mice was comparable, qualitative differences in the pattern of demyelination were observed in TLR2 / mice. Yet, spinal TNF-a and iNOS levels were lower in the injured TLR2 / mice than in the injured WT mice. It was therefore postulated that TLR2 and TLR4 play neuroprotective roles in SCI even though the underlying mechanisms for this beneficial effect remain undefined (Kigerl et al., 2007). It is worth noting that developmental TLR2 and TLR4 deficiency in the whole-animal mutant or knockout mice can alter baseline immune and CNS function which could be a confounding factor when the roles of TLRs in SCI are analyzed. Therefore, it would be valuable to determine whether antagonist-mediated blockade of TLR2 or TLR4 in SCI-sustaining WT mice leads to functional deficits and histopathological alterations similar to those observed in TLR4 mutant and TLR2 / mice. Although the effects of antagonist mediated-blockade of TLR4 versus TLR4 deficiency in mutant or knockout mice have not been compared in the context of SCI, they have been evaluated in studies of the brain. When memory function was assessed, inhibition of TLR4 in the CNS of the WT mice and developmental TLR4 deficiency in the mutant mice resulted in different behavioral outcomes (Okun et al., 2012). While investigations on whole body TLR mutant or knockout mice can provide valuable information, these latter investigations highlight the importance of complementing or corroborating such studies by use of TLR ligands.

4.1. Expression of TLRs in the injured spinal cord

4.3. Effects of TLR ligands in spinal cord injury

TLRs play an important role in mediating the inflammatory response in the spinal cord. Injury to the mouse spinal cord evokes

A recent report by Stirling et al. (2014) used an unconventional, ex-vivo, laser-induced SCI model in order to investigate the effects

4. TLRs in spinal cord injury

Please cite this article in press as: Heiman, A., et al. Toll-like receptors in central nervous system injury and disease: A focus on the spinal cord. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.06.203

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of the TLR2 agonist PAM2CSK4 on spinal cord microglia. This ex-vivo SCI model was purposely used to analyze the responses of resident microglia in the absence of infiltrating blood-derived myeloid cells and to determine whether the M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes, described in monocytes and peripheral macrophages, are also observed in resident CNS microglia. Perfusion of the isolated spinal cord with PAM2CSK4 was initiated before induction of injury and continued until 4 h post-injury. PAM2CSK4 increased the microglial response, modified the inflammatory milieu by augmenting both M1 and M2 markers and effectors, and reduced secondary loss of myelinated axons. Although the authors did not find a clear M1 or M2 phenotype in the microglia, PAM2CSK4 induced an alternative microglial activation profile that displayed a mixture of M1 and M2 characteristics. While the studies could not determine the exact causes of PAM2CSK4-induced axonal protection, the authors cautioned about the use of anti-inflammatory treatments that target microglial activation in SCI. Interestingly, neuroprotection was not observed when zymosan, a different TLR2 agonist, was used (Stirling et al., 2014). In the study of Stirling et al. (2014), not only microglia but also additional cell types that express TLR2, could have contributed to the neuroprotective effects of PAM2CSK4. In addition, the divergent results obtained with zymosan and PAM2CSK4, could be due to the TLR2-containing signaling complex engaged by each agonist as TLR2/TLR1 and TLR2/TLR6 heterodimers have been previously described by Ozinsky et al. (2000b). With regard to TLR4, Guth et al. (1994) administered the agonist LPS systemically (intraperitoneal, i.p.) to rats sustaining a crush SCI and found that it reduced cavitation without altering lesion size and appeared to enhance the preservation of axons. In addition, daily i.p. injections of LPS to rats sustaining a moderate contusion injury improved recovery of locomotor function as assessed by the Tarlov scale; however, locomotor recovery was not observed when the injury was severe (Guth et al., 1994). In another study (Vallieres et al., 2006), daily i.p. LPS injections increased the number of activated microglia/macrophages in regions that contained tracts undergoing Wallerian degeneration and enhanced the phagocytosis of degenerating myelin in mice sustaining a hemi-section injury to the spinal cord. In contrast to the investigations on the effects of LPS in the naïve spinal cord, the studies of Guth et al. (1994) and Vallieres et al. (2006) support the idea that LPS could help recovery of the injured spinal cord by inducing neuroprotection or by enhancing the removal of myelin debris, which is known to contain components that inhibit axonal repair. These discrepant outcomes might be partly due to the route of LPS administration: systemic delivery in SCI studies versus i.t. or intraspinal delivery in investigations on the naïve spinal cord. Systemic LPS treatment could impact the outcome of SCI by altering the peripheral immune response to SCI. In addition, the milieu in the injured versus naïve spinal cord is different and could react to LPS in a distinct manner. Toll like receptor 9 plays a role in modulating inflammation and the functional outcomes of SCI. Intrathecal administration of a TLR9 antagonist, CpG ODN 2088, to mice sustaining a severe mid-thoracic spinal cord contusion injury reduced the number of cells of myeloid and hematopoietic origin within the epicenter, decreased TNF-a mRNA expression and ameliorated heat hypersensitivity (David et al., 2013). In addition, intrathecal delivery of CpG ODN 2088 improved white matter sparing and injury-induced bladder dysfunction (David et al., 2014). It is possible that CpG ODN 2088 exerts its anti-inflammatory effects by interfering with the binding of endogenous DAMPs to TLR9. The mechanisms underlying the effects of the TLR9 antagonist on heat hypersensitivity are currently under investigation.

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4.4. Expression of endogenous TLR ligands following spinal cord injury Endogenous ligands of TLRs are released from necrotic and stressed cells or inflammatory cells at the sites of the SCI. These ligands include hyaluronan (Struve et al., 2005), heat shock protein 70 (HSP70) (Mautes and Noble, 2000), HSP60 (Lehnardt et al., 2008), HMGB-1 (Asea et al., 2002; Yu et al., 2006), mRNA (Asea et al., 2002; Ohashi et al., 2000) and aB-crystallin (van Noort et al., 2013). HMGB-1 is a DNA binding protein that regulates gene transcription. In pathological conditions, HMGB-1 is passively released by damaged or necrotic cells or actively released by activated inflammatory cells (Scaffidi et al., 2002). HMGB-1 can bind both TLR2 and TLR4 (Yu et al., 2006). Following a mid-thoracic compression injury in the rat, the expression of HMGB-1 is transiently upregulated primarily in neurons and macrophages but not astrocytes (Chen et al., 2011). This SCI-elicited increase in HMGB-1 occurs as early as 6 h post injury (p.i.), peaks at 3 days p.i. and is no longer observed by 2 weeks p.i. TLR2 and TLR4 expression are also upregulated at 3 days p.i., but not at any other time point analyzed (Chen et al., 2011). Whereas these studies appeared to suggest that HMGB-1 could modulate the inflammatory response, at least partly via binding TLR2 and TLR4, its precise role in SCI remains to be determined. aB-crystallin (HspB5) is a member of the heat shock protein family and has anti-apoptotic, neuroprotective and anti-inflammatory effects (Ousman et al., 2007). Van Noort et al. (2013) have shown that aB-crystallin containing microparticles activate an immune regulatory response in peripheral macrophages which is dependent on TLR1, TLR2 and CD14. Knockdown of CD14 and TLR1/2 but not other TLRs reduces HspB5-induced release of TNF-a by the human macrophage-like cell line THP-1 (van Noort et al., 2013). In the uninjured mouse spinal cord, aB-crystallin is expressed constitutively by oligodendrocytes and is absent in astrocytes (Klopstein et al., 2012). Following a spinal cord contusion injury, aB-crystallin was localized to both oligodendrocytes and astrocytes but not other cells including microglia and neurons. aB-crystallin expression in the spinal cord was decreased following SCI and intravenous administration of human aB-crystallin improved open field locomotor function and tissue sparing, promoted NF-jB expression and increased chemokine and cytokine levels (Klopstein et al., 2012). 5. TLRs and neuropathic pain Neuropathic pain often accompanies injuries and diseases of the nervous system and can be categorized as either peripheral or central. Generally, injury to and/or inflammation of the sensory nerves are sufficient to induce neuropathic pain (reviewed in Koltzenburg and Scadding, 2001). Peripheral neuropathic pain can result from lesions to the peripheral nervous system (PNS) elicited by mechanical trauma (Bennett and Xie, 1988; Malmberg and Basbaum, 1998; Seltzer et al., 1990), exposure to chemicals (Bandell et al., 2004; Caterina et al., 1997; McKemy et al., 2002; Peier et al., 2002), infection (Takasaki et al., 2000), and/or tumor invasion (Schwei et al., 1999) and involves pathophysiological changes within both the PNS and CNS (reviewed in Dworkin et al., 2003a). As current therapies do not adequately alleviate neuropathic pain, new targets for therapeutic interventions, including TLRs, are under investigation. 5.1. Overview of molecular mechanisms that mediate pain responses in the spinal cord A variety of mechanisms in the spinal cord mediate neuropathic pain responses (for a comprehensive review see Basbaum et al.,

Please cite this article in press as: Heiman, A., et al. Toll-like receptors in central nervous system injury and disease: A focus on the spinal cord. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.06.203

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2009). Hyperexcitation of dorsal horn neurons that convey sensory information from the spinal cord to the brain is one of the factors that drive the development of neuropathic pain. This hyperexcitability, referred to as central sensitization (Woolf, 1983), is the consequence of molecular changes in the spinal cord dorsal horns including a more pronounced glutamatergic excitation (Seltzer et al., 1991; Kawamata and Omote, 1996) and an attenuated GABAergic inhibition (Moore et al., 2002; Drew et al., 2004). Following peripheral nerve injury and SCI, increased glutamate release by stressed or dying cells (Newcomb et al., 1997) and activated microglia (Gwak and Hulsebosch, 2009; Hains and Waxman, 2006) results in the accumulation of glutamate in the extracellular milieu. In addition, reduced glutamate uptake (Binns et al., 2005) due to the downregulation of the astroglial glutamate–aspartate transporter (GLAST) (Sung et al., 2003), also known as excitatory amino acid transporter 1 (EAAT1), and glutamate transporter 1 (GLT-1/EAAT2; Lepore et al., 2011a,b; Olsen et al., 2010) contribute to the rise in the extracellular glutamate concentrations. A decrease in GABA and glycine inhibition facilitates the excitability of dorsal horn neurons. The death of GABAergic interneurons (Drew et al., 2004; Meisner et al., 2010; Moore et al., 2002) in the dorsal horn weakens GABAergic tone. Moreover, the increased transport of GABA from the extracellular environment, as a consequence of increased GABA transporter GAT1 (Daemen et al., 2008) expression, further reduces GABAergic inhibition. In addition, changes in the density of descending serotoninergic afferents rostral and caudal to the lesion have been implicated in the modulation of neuropathic pain following SCI (Bruce et al., 2002; Oatway et al., 2004) whereas a loss in descending noradrenergic inhibition was associated with peripheral nerve injury-elicited pain (Rahman et al., 2008). Chronic activation of microglia (Popovich et al., 1997; Sroga et al., 2003; Detloff et al., 2008) and astrocytes (Garrison et al., 1991; Coyle, 1998), as well as peripheral immune cells infiltrating the spinal cord are critical contributors to neuropathic pain mechanisms, via the release of effectors including TNF-a (Cunha et al., 1992), IL-1b (Ferreira et al., 1988), IL-6 (Arruda et al., 1998), ATP (Zhou et al., 2001), brain derived neurotrophic factor (BDNF) (Coull et al., 2005), and nerve growth factor (NGF) (Lewin et al., 1994; Woolf et al., 1994). The investigations on mechanisms underlying nerve injuryinduced neuropathic pain have been summarized in comprehensive reviews (Austin and Moalem-Taylor, 2010; Nicotra et al., 2012). Here we will highlight the findings of only a few reports showing the dependence of peripheral nerve injury-induced neuropathic pain on TLRs. Subsequently, we will discuss the contribution of TLRs to central neuropathic pain. 5.2. Toll-like receptors in peripheral nerve injury-induced neuropathic pain Rodent models for peripheral neuropathic pain include chronic constriction injury (CCI) of the sciatic nerve (Bennett and Xie, 1988) and partial sciatic nerve ligation (SNL; Seltzer et al., 1990), as well as complete sciatic nerve transection (SNT; Sugimoto et al., 1990). Most studies focus on stimulus-induced/peripherally-evoked pain that can be elicited in response to either normally nonnoxious (allodynia) or noxious (hyperalgesia) stimuli which are tested by evaluating mechanical or thermal paw withdrawal thresholds (for reviews see Barrot, 2012; Gregory et al., 2013). Toll like receptor 2 and TLR4 are the most widely studied PRRs in the context of peripheral nerve injury-induced neuropathic pain, although investigations on other TLRs have been reported. A lumbar fifth (L5) nerve transection model in WT and TLR2 / mice and in vitro approaches were used to demonstrate the role of TLR2 in the production of inflammatory mediators by spinal cord glia

in vitro as well as the induction of mechanical allodynia and thermal hyperalgesia (Kim et al., 2007). Primary mixed spinal cord glia, obtained from WT mice, responded to the supernatant of a sensory neuron cell line lysate (used as stimuli from damaged neurons) by increasing the mRNA expression of TNF-a, IL-1b and IL-6. This response was abolished in spinal glia obtained from TLR2 / mice. Similarly, nerve injury-induced increase in the expression of these inflammatory mediators was lower in the spinal cord of TLR2 / mice than WT controls. Both mechanical allodynia and thermal hyperalgesia were attenuated in the TLR2 / mice. These findings, taken together, suggested that spinal TLR2 modulates pain responses via release of inflammatory mediators from glial cells (Kim et al., 2007). The dependence of neuropathic pain development on TLR2 has been confirmed in a partial SNL model in WT and TLR2 / mice, even though TLR2 deficiency only partially reduced mechanical allodynia whereas it abolished thermal hypersensitivity entirely (Shi et al., 2011b). However, in contrast to the results of the Kim et al. (2007) study, this latter investigation attributed the functional deficits to TLR2-dependent molecular changes occurring in the damaged nerve rather than the spinal cord. Increased TLR2 mRNA expression was observed in the macrophages of the damaged WT nerve, which was paralleled by increased TNF-a expression. Both macrophage recruitment and TNF-a expression were significantly lower in the damaged sciatic nerve of the TLR2 / mice as compared to the WT, supporting the idea that macrophage recruitment and cytokine expression are dependent, at least partly, on TLR2. On the contrary, TLR2 expression was not observed in the spinal cord following WT nerve ligation and TLR2 deficiency did not impact microglia activation or TNF-a expression in the spinal cord of WT mice (Shi et al., 2011b). Subsequent reports supported the notion that was put forward by Kim et al. (2007) which highlighted TLR2-dependent changes in the spinal cord following peripheral nerve injury (Freria et al., 2012; Lim et al., 2013). It is worth noting that all studies showing TLR2-dependent changes in the spinal cord were performed on animal models of nerve transection injury whereas the study that did not show TLR2-dependent changes in the spinal cord (Shi et al., 2011b) utilized a nerve ligation model. It is possible that these discrepant results are due to differences in the injury models utilized in the various investigations. The contribution of TLR4 to nerve injury-induced neuropathic pain has been extensively studied. Tanga et al. (2005) showed that the development of neuropathic pain is dependent on TLR4. A subsequent study by Hutchinson et al. (2008) indicated that chronic i.t. infusion of the non-stereoselective blockers of TLR4 signaling, (+)naloxone or ( )naloxone, to rats sustaining a sciatic nerve CCI, reversed well-established mechanical allodynia. These findings support the notion that TLR4 participates in both the initiation and maintenance of neuropathic pain. In agreement with this idea, Lewis et al. (2012) reported that subcutaneous injections of (+)naloxone reduced both acute and long-established pain in a rat SNL model; (+)naloxone exerted its beneficial effects even when administered as late as 4 months post-SNL. In a rat sciatic nerve CCI model, siRNA-mediated knockdown of TLR4 expression reduced injury-induced thermal hypersensitivity and tactile allodynia, which was paralleled by inhibition of NF-jB p65 activation as well as TNF-a and IL-1b production in the lumbosacral spinal cord (Wu et al., 2010). Additionally, i.t. injection of the TLR4 inhibitor, epigallocatechin gallate (EGCG), to rats sustaining a CCI of the sciatic nerve restored the paw withdrawal latency in response to both thermal and mechanical stimuli (Kuang et al., 2012). This was accompanied by a significant reduction in TLR4 and HMGB-1 transcript and protein levels, a decrease in TNF-a and IL-1b as well as an increase in IL-10 protein levels in the lumbar spinal cord as compared to injured rats receiving saline (Kuang et al., 2012).

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The roles of TLRs 2, 3, 4, and 5 have been studied in their respective knockout mice sustaining an L5 SNL (Stokes et al., 2013a). Peripheral nerve injury resulted in the development of ipsilateral mechanical allodynia in both WT and knockout mice. However, injured male mice deficient in TLRs 2, 3, 4, or 5 exhibited a significant, albeit partial, reduction in tactile allodynia as compared to their WT counterparts. As MyD88 mutant mice also showed partial reversal of tactile allodynia, it was concluded that mechanisms other than those mediated by TLRs contribute to the pain responses. It is worth noting that tactile allodynia was attenuated only in male, but not the female TLR4 knockout mice, indicating a sex difference in TLR4-mediated pain mechanisms (Stokes et al., 2013a). 5.3. Toll like receptors in central neuropathic pain Several studies have shown that i.t. administration of TLR agonists to naïve rodents elicits transient neuropathic pain. Intrathecal injection of LPS to naïve rats increased thermal hyperalgesia without changing mechanical withdrawal thresholds (Meller et al., 1994). Subsequent investigations indicated that i.t. administration of LPS enhances the activity of spinal cord dorsal horn neurons (Reeve et al., 2000). The same report showed that similar effects are observed following i.t. delivery of IL-1b and TNF-a, even though the effects of IL-1b were of a larger magnitude than those of TNF-a. Intrathecal IL-1b, but not TNF-a, also induced mechanical allodynia and hyperalgesia (Reeve et al., 2000). The studies of Clark et al. (2006) indicated that i.t. administration of LPS promotes mechanical hyperalgesia and tactile allodynia. A single i.t. LPS injection was not sufficient to induce a pain response and two consecutive i.t. injections, performed 24 h apart, were necessary to elicit mechanical hyperalgesia and tactile allodynia (Clark et al., 2006). The release of IL-1b by activated microglia appeared to mediate the pain response to LPS and was dependent on the presence or activity of the purinergic receptor P2X7 (Clark et al., 2010). In contrast, Saito et al. (2010) reported induction of tactile allodynia following a single i.t. LPS injection, although this response was transient and short-lived. In this study, LPS elicited the release of prostaglandin E(2) and TNF-a, which was blocked by inhibitors of microglial and astroglial activation. The investigations of Hutchinson et al. (2009) failed to show an effect of a wide range of LPS doses on mechanical allodynia when the TLR4 agonist was delivered as a single i.t. injection to naïve rats. However, mechanical allodynia was induced by a combination of LPS and dimethylsulfoxide (DMSO). This effect was attributed to DMSO-mediated release of heat shock protein 90 (HSP90) (Hutchinson et al., 2009). HSP90 has been previously shown to act as a co-factor and to form clusters with TLR4 within lipid microdomains upon LPS stimulation (Triantafilou and Triantafilou, 2004). Further studies indicated that TLR4 is required, but not sufficient, to induce central pain and cooperation by other components of the TLR4 receptor complex is necessary to elicit pain responses (Hutchinson et al., 2009). In addition to spinal TLR4, CD14, the co-receptor for TLR2 and TLR4, also contributes to neuropathic pain mechanisms in the spinal cord (Cao et al., 2009). Intraspinal administration of a soluble mouse CD14-human IgG Fc fusion protein increased tactile sensitivity within one hour post-injection. The allodynic response was much stronger in WT than TLR4 mutant mice although it was not completely abolished in the absence of functional TLR4. Therefore, it is likely that CD14 acts in conjunction with additional TLRs to mediate pain responses (Cao et al., 2009). Interestingly, a significant sex difference in mechanical allodynia elicited by i.t. LPS was identified. Male, but not female, mice exhibited an allodynic response (Sorge et al., 2011). Furthermore, this sex difference in the TLR4-mediated pain response appeared

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to be confined to the spinal cord as cerebral intraventricular administration of LPS to male and female mice evoked similar responses. In addition, the sex difference was observed in pain mechanisms but not other functional parameters. In contrast to TLR4, zymosan-elicited allodynia did not reveal any sex differences. Investigations on gonadectomized male and female mice, with and without testosterone replacement, indicated that this gender difference is mediated by testosterone. It was suggested that, in the absence of testosterone, female mice utilize a TLR4independent pain-processing pathway and administration of testosterone to gonadectomized females facilitates a switch in pain processing from a TLR4-independent to a TLR4-dependent pathway (Sorge et al., 2011). Other TLRs have also been implicated in the mediation of central neuropathic pain. Stimulation of spinal TLR3 by i.t. administration of poly I:C to male rats resulted in the development of mechanical allodynia which was followed by microglial activation (Mei et al., 2011). The investigations of Liu et al. (2012) suggested that TLR3 is critical for central sensitization-driven pain hypersensitivity. In this study, the basal thermal and mechanical sensitivity of TLR3 deficient and WT mice were comparable. In addition, acute spontaneous pain, elicited by the intraplantar injection of capsaicin and mustard oil were similar in both groups. However, intraplantar injection of formalin induced a biphasic pain response with an early phase that corresponds to activation of peripheral nociceptors and a second phase attributed to central sensitization. There were no significant differences between the pain responses observed in TLR3 deficient and WT mice during the first phase whereas pain responses were significantly attenuated in TLR3 deficient mice during the second phase. Therefore, it was concluded that TLR3 does not play a role in acute pain but is critical for pain hypersensitivity which is dependent on central sensitization (Liu et al., 2012). As indicated above, our laboratory has reported that in female mice sustaining a severe thoracic spinal cord contusion injury, blockade of TLR9 via i.t. delivery of a TLR9 antagonist, CpG ODN 2088, reduces thermal hypersensitivity as compared to vehicletreated injured mice (David et al., 2013). As the inflammatory response was attenuated by the treatment, we postulated that CpG ODN 2088 alleviates pain responses most likely by reducing the TLR9-mediated pro-inflammatory reaction in the spinal cord. However, since TLR9 is expressed in spinal cord neurons (David et al., 2013), a direct effect of the antagonist on neurons of the dorsal horn, which convey sensory information to the brain, could not be ruled out. This issue is currently under investigation in our laboratory. Although tactile allodynia can be induced by i.t. administration of different TLR agonists, Stokes et al. (2013a,b) investigated the extent and duration of the allodynic response and the signaling mechanisms mediating induction of the pain state, and found that they appear to vary depending on the TLR. Intrathecal administration of heat killed Listeria monocytogenes (HKLM, a TLR2 agonist), flagellin (FLA-ST, a TLR5 ligand), poly (I:C) and LPS induced tactile allodynia. However, the response to flagellin lasted only 3 h while the response to all other TLR ligands lasted for 7 days. The TLR2 and TLR4 ligand-elicited tactile allodynia was TNF-a dependent and was mediated via the TRIP-containing signaling cascade as indicated by the abrogation of the pain response in TRIP-deficient mice. In contrast, TLR3 ligand-elicited tactile allodynia was TNF-a independent and involved the TRIF-containing signaling cascade. Interestingly, the TRIF deficient mice exhibited prolonged allodynia in response to LPS which appeared to be due to the lack of IFN-b. Thus, activation of TLR4 can induce allodynia via the TRIP pathway and production of TNF-a but can also inhibit allodynia via the TRIF pathway and production of IFNb (Stokes et al., 2013a,b).

Please cite this article in press as: Heiman, A., et al. Toll-like receptors in central nervous system injury and disease: A focus on the spinal cord. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.06.203

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6. TLRs in amyotrophic lateral sclerosis (ALS) and mouse models of ALS Amyotrophic lateral sclerosis is a fatal, neurodegenerative, adult onset motor neuron disease characterized by progressive motor neuron loss in the spinal cord, cortex and brainstem. Neurodegeneration is paralleled by CNS inflammation and glial activation (for reviews see Bowerman et al., 2013; Turner et al., 2013; Valori et al., 2014). Both genetic and environmental factors appear to contribute to ALS pathogenesis (Renton et al., 2014). Approximately 5–10% of all ALS cases are inherited and collectively named familial ALS (fALS) whereas about 90–95% of all ALS cases have no family history and are referred to as sporadic ALS (sALS). Missense mutations in the gene that encodes Cu/Zn superoxide dismutase (SOD1) are one of the most frequently observed and best-studied contributors to fALS (Rainero et al., 1994; Rosen, 1993; Tsuda et al., 1994) although other mutations have also been implicated in fALS (Neumann et al., 2006; Kwiatkowski et al., 2009; Vance et al., 2009). Transgenic mice that express various mutated forms of human SOD1 have been created and used as animal models of ALS (reviewed in Bruijn et al., 2004; Van Den Bosch, 2011). In general, these mice exhibit a non-symptomatic phase which is followed by a symptomatic phase during which the disease progresses. Evidence indicates that both glia (Beers et al., 2006; Hall et al., 1998; Liu et al., 2009b; Nagai et al., 2007) and neurons (Boillee et al., 2006; Clement et al., 2003) expressing mutant SOD1 contribute to the onset and progression of the disease. Studies have shown that aggregates of the misfolded mutant SOD1 within neurons (Johnston et al., 2000) and secreted mutant SOD1 (Turner et al., 2005; Urushitani et al., 2006) promote neuronal death, the latter via induction of inflammation. There are also mice with spontaneous mutations in different genes, which mimic human motor neuron disease. Among these, the wobbler mouse is the best characterized and has been used most frequently as a model of motor neuron disease (Andrews et al., 1974). A point mutation in the Vsp54 subunit of the Golgiassociated retrograde protein complex has been associated with motor neuron disease in the wobbler mouse (Schmitt-John et al., 2005). An asymptomatic phase until 3 weeks of age is followed by a phase during which clinical symptoms begin to appear. Clinical symptoms progress from 3 weeks until 3 months of age and significant degeneration of motor neurons occur (Boillee et al., 2003). TLRs have been implicated in mediating sterile inflammation associated with ALS. The presence of TLR2 and CD14 immunoreactivity in infiltrating perivascular but not parenchymal mononuclear phagocytes in the spinal cord of ALS patients has been described (Letiembre et al., 2009). Subsequent studies by Casula et al. (2011) showed increases in TLR2 and TLR4 mRNA levels in the post-mortem spinal cord of sALS patients. Analyses of cellular distribution by immunocytochemistry and in situ hybridization localized TLR2 to cells of the microglia/macrophage lineage, but not reactive astrocytes, in both the white and gray matter. In contrast, TLR4 mRNA and immunoreactivity were found in the residual motor neurons of the ventral horn and in reactive astroglia but were absent in cells of the microglia/macrophage lineage. mRNA expression levels of HMGB-1 were also upregulated in the ALS spinal cord in both microglia and astrocytes (Casula et al., 2011). Whereas these studies suggest a role for TLR2 and TLR4 in ALS, their precise contribution remains undefined. However, in vitro investigations unravel potential mechanisms. Liu et al. (2009b) determined that the release of TNF-a by BV2 microglial cell line overexpressing mutant human SOD1 was more pronounced than BV2 microglia expressing WT human SOD1, following stimulation of TLR2. This heightened release of TNF-a was dependent on the generation of reactive oxygen species (ROS) and activation of

ROS-sensitive TNF-a converting enzymes. Based on these findings it was suggested that TLR2 stimulation increases the neurotoxicity of mutant human SOD1-expressing microglia by enhancing ROSmediated release of TNF-a (Liu et al., 2009b). However, it remains to be determined whether this hypothesis, which was based on findings with a microglia cell line, holds true in primary microglia, in animal models of ALS and in the human disease. Other studies have explored how stimulation of innate immunity affects the outcomes of motor neuron disease in mouse models of ALS. Repeated, systemic administration of LPS to presymptomatic SOD1 transgenic mice exacerbated disease progression and shortened life span, which was associated with a more severe loss of motor neuron axons, degeneration of motor neurons and induction of TLR2 expression in parenchymal microglia found in regions of neuronal and astroglial death in the ventral lumbar (L5) spinal cord (Nguyen et al., 2004). This study hypothesized that microglia are the likely mediators of toxicity in regions of cell degeneration. In agreement with this notion, the studies of Zhao et al. (2010) indicated that extracellular mSOD1 induces microglial activation, increases the release of effectors including TNF-a and IL-1b and promotes expression of iNOS as compared to SOD1WT-treated microglia, in vitro. Additionally, when primary motor neurons were co-cultured with microglia in the presence of mSOD1 or wild type SOD1, motor neuron loss was observed in cultures treated with mSOD1. This toxic effect was mediated by microglia since direct exposure of motor neurons to mSOD1, in the absence of microglia, did not induce death of motor neurons (Zhao et al., 2010). In the wobbler mouse, systemic, chronic administration of a TLR4 antagonist, VB3323, during the symptomatic phase improved behavioral outcomes including grip strength, and reduced chromatolysis, glial activation and TNF-a production in the cervical spinal cord (De Paola et al., 2012). It is not yet known whether the beneficial effects of the TLR4 antagonist are mediated by direct actions on spinal cord cells, or indirectly via systemic responses.

7. Discussion The investigations in animal models of SCI, neuropathic pain and ALS underscore the importance of unraveling the pivotal role of TLRs in spinal cord pathology. These studies highlight the need for further research in this field. A number of potential future directions are summarized in Table 1 and discussed below. Undoubtedly, understanding the contribution of TLRs to spinal cord pathology is a complex and challenging task which is compounded by the fact that all glial subtypes and neurons, as well as immune system-related cells that infiltrate the spinal cord in injury and disease, express TLRs and respond to TLR activation in distinct manners. Illustrating this issue, activation of neuronal TLR3 results in growth cone collapse suggesting that TLR3 signaling could constitute an impediment to axonal regeneration (Cameron et al., 2007), whereas human astrocytes, stimulated with a TLR3 agonist, secrete neurotrophic factors including BDNF, neurotrophin 4 (NT-4) and CNTF (Bsibsi et al., 2006), raising the possibility of TLR3-mediated neuroprotection. Thus, beneficial as well as detrimental effects could occur simultaneously and the net outcome of TLR activation could depend on the way the responses of the various cell types are integrated. It is also likely that different TLRs are involved simultaneously in spinal cord pathology with some TLRs exerting opposite effects, others cooperating with each other or modulating the function of each other (Butchi et al., 2010), as seen in infection or autoimmunity (Desnues et al., 2014; Moody et al., 2014). Therefore, preclinical studies that aim to test the potential of TLR ligands as therapeutic agents should take into consideration the crosstalk

Please cite this article in press as: Heiman, A., et al. Toll-like receptors in central nervous system injury and disease: A focus on the spinal cord. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.06.203

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Table 1 Some future directions that could improve the understanding of the role of TLRs in spinal cord pathology.  Development of neuron and glial subtype specific conditional knockout mice to elucidate the role of TLRs in specific cell types, in vivo, under physiological and pathological conditions of the spinal cord  Corroboration of the results obtained with whole body knockout or mutant mice by use of TLR antagonists in animal models of SCI and neuropathic pain  Further analysis of intracellular TLRs and their ligands in SCI and neuropathic pain  Elucidation of the interactions between different TLRs in spinal cord pathology  Elucidation of the potential cross-talk between TLRs and other PRRs in spinal cord pathology  Unraveling the effects of distinct agonists or antagonists of a specific TLR in order to define ligand-dependent outcomes in spinal cord pathology  Identification of novel endogenous ligands of TLRs and comparison of their effects with synthetic agonists that are often used to activate TLRs in experimental models of spinal cord pathology

between different TLRs and evaluate combinatorial strategies that use concurrent or sequential administration of TLR agonists or antagonists. It is also important to note that different ligands for a specific TLR could have diverging effects. Such ligand-selective effects could be dependent on the co-receptor with which the TLR associates and the formation of heterodimers between different TLRs. In addition, it is essential to identify the endogenous ligands of TLRs and characterize more systematically their mode of action in the spinal cord. The majority of the investigations on TLRs use synthetic ligands or bacteria-derived activators, which might exert actions dissimilar to those induced by endogenous ligands. In future studies, the use of endogenous ligands could provide more accurate insights into TLR-mediated cellular responses that occur in injury and disease. Finally, most of the current literature regarding the role of TLRs in spinal cord pathology focuses on glia- and especially, macrophage-mediated responses. However, neurons also express TLRs and could respond directly to TLR activation or inhibition. This particular aspect has been understudied in the context of spinal cord pathology and warrants further investigations. Despite this complexity, a number of commonalties have emerged from studies that investigated the role of TLRs in the naïve and injured spinal cord and in neuropathic pain. The bestcharacterized TLRs in the context of SCI and pain mechanisms are TLR2 and TLR4. Both neurotoxic and neuroprotective or repair-promoting roles have been attributed to these receptors in spinal cord pathology and it is likely that the beneficial and detrimental effects occur concurrently, partly via actions on local glia or infiltrating cells, especially macrophages. As stated above, the opposing effects of TLR2 and TLR4 may be dependent on the ligand that activates the receptors, the co-receptors that associate with the TLR, the signaling mechanisms that result in release of different effectors, the cell types that are engaged, and the milieu in which these events occur. Therefore, the global outcome of TLR2 or TLR4 activation could reflect the most dominant effect under specific circumstances. In the face of such opposing effects, the use of TLR2 and TLR4 agonists or antagonists as therapeutic agents in spinal cord pathology might be a challenging task. Information regarding the contribution of intracellular TLRs to SCI or neuropathic pain is more limited. Nevertheless, emerging evidence suggests that both TLR3 and TLR9 are promising candidates and should be further analyzed. The modulation of pain responses by TLR3 ligands highlights the importance of this receptor in spinal mechanisms mediating nociception, whereas inhibition of TLR9 in SCI might confer neuroprotection via regulation of sterile inflammation and could prevent the development of neuropathic pain. However, it remains to be determined whether TLR9 antagonists can ameliorate well-established neuropathic pain, a finding that would increase the value of these antagonists as potential therapies. Currently, treatments for central neuropathic pain are largely restricted to the use of opiates (Rowbotham et al., 2003), gabapentin (Moore et al., 2011), or pregabalin (Dworkin et al., 2003b; Freynhagen et al., 2005). However, dorsal horn neurons are less responsive to inhibition by l-opioid agonists (Kohno

et al., 2005). Moreover, this treatment ameliorates pain only in some patients experiencing neuropathic pain. Therefore, uncovering more effective treatments and defining new targets for interventions are important goals. In this context, TLR ligands show the potential of being likely candidates. With regard to TLRs in ALS, the field is still in its infancy. Most of the current information is based on results obtained in vitro and these findings need to be further corroborated in vivo. In in vivo investigations on animal models of ALS, TLR ligands were administered systemically. As the treatment was not confined to the CNS, the effects could reflect changes in systemic immune responses as well as the CNS. Studies that use i.t. delivery of TLR ligands in animal models of ALS could define the role of these receptors in the onset, development or progression of the disease as it pertains to mechanisms occurring in the spinal cord. In addition, most studies focused on TLR2 and TLR4 and additional information is necessary regarding the contribution of the other TLRs. When considering TLR ligands as potential therapeutic agents for spinal cord pathology, it is necessary to take into account that TLR activation induces systemic immune responses. Hence, to rule out undesired adverse effects, it is critical to monitor immune responses even when the route or administration is i.t. Moreover, because some TLR ligands have limited ability to cross the blood–spinal cord barrier and because systemic delivery of TLR ligands could have a global impact on immune system function in addition to CNS function, i.t. administration could be a preferred route of delivery. Osmotic pumps for the i.t. delivery of various agents have been used in the clinical setting (Saval and Chiodo, 2010) and could be adapted for the administration of TLR ligands, even though this approach is more invasive, requires surgery, and has the risk of late mechanical failures or infections related to the device which delivers the medication (the pump system). In summary, a growing body of evidence supports the notion that TLRs are key modulators of sterile inflammation and glial activation associated with SCI, although it remains to be determined whether they mediate neuroprotection or neurodegeneration via direct effects on spinal neurons. These receptors are also key players in central neuropathic pain mechanisms occurring in the spinal cord albeit the overlapping, opposing or divergent roles warrant further investigations. Further identification of endogenous ligands of TLRs and elucidation of their effects in the spinal cord could advance our understanding of TLR-mediated physiological and pathological processes in the spinal cord. Additional studies are required to define the effectiveness of TLR ligands as therapeutic agents in spinal cord injury and disease.

Acknowledgments Supported by New Jersey Commission on Spinal Cord Research grant CSCR12IRG007 and Reynolds Family Spine Laboratory Funds. This review was conceived as a concise overview of investigations on TLR in spinal cord pathology. We apologize to those who published excellent reports in this broad field but were not cited here.

Please cite this article in press as: Heiman, A., et al. Toll-like receptors in central nervous system injury and disease: A focus on the spinal cord. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.06.203

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Please cite this article in press as: Heiman, A., et al. Toll-like receptors in central nervous system injury and disease: A focus on the spinal cord. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.06.203

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Please cite this article in press as: Heiman, A., et al. Toll-like receptors in central nervous system injury and disease: A focus on the spinal cord. Brain Behav. Immun. (2014), http://dx.doi.org/10.1016/j.bbi.2014.06.203